Abstract

The human AmphyphisinII/Bin1 N-BAR domain belongs to the BAR domain superfamily, whose members sense and generate membrane curvatures. The N-BAR domain is a 57 kDa homodimeric protein comprising a six helix bundle. Here we report the protein folding mechanism of this protein as a representative of this protein superfamily. The concentration dependent thermodynamic stability was studied by urea equilibrium transition curves followed by fluorescence and far-UV CD spectroscopy. Kinetic unfolding and refolding experiments, including rapid double and triple mixing techniques, allowed to unravel the complex folding behavior of N-BAR. The equilibrium unfolding transition curve can be described by a two-state process, while the folding kinetics show four refolding phases, an additional burst reaction and two unfolding phases. All fast refolding phases show a rollover in the chevron plot but only one of these phases depends on the protein concentration reporting the dimerization step. Secondary structure formation occurs during the three fast refolding phases. The slowest phase can be assigned to a proline isomerization. All kinetic experiments were also followed by fluorescence anisotropy detection to verify the assignment of the dimerization step to the respective folding phase. Based on these experiments we propose for N-BAR two parallel folding pathways towards the homodimeric native state depending on the proline conformation in the unfolded state.

Highlights

  • Understanding of the folding mechanism of proteins is a key challenge in molecular biophysics

  • In the present study we investigated the equilibrium and kinetics of urea induced un- and refolding of the AmphiphysinII/Bin1 N-BAR domain by fluorescence, fluorescence anisotropy and far-UV circular dichroism (CD) spectroscopy

  • Because of the dimeric character of N-BAR the apparent thermodynamic stability monitored by equilibrium unfolding transition curves depends on the protein concentration

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Summary

Introduction

Understanding of the folding mechanism of proteins is a key challenge in molecular biophysics. FIS and H2A/H2B show such a fast association of the monomers near the diffusion limit followed by a slower folding step to build the native dimer[14,15] These issues have to be taken into account compared to monomeric proteins. BAR domains with an amphipathic helix (helix0) at their N-terminus are called N-BAR and exhibit a higher activity during tubulation These helices are only stable in the presence of lipids, otherwise they are unstructured[29]. We provide a detailed study on the folding mechanism of the homodimeric N-BAR domain by combining various biophysical methods including intrinsic tryptophan fluorescence, fluorescence anisotropy and CD spectroscopy. Urea equilibrium unfolding transitions show no intermediates, whereas kinetic single- and double-mixing experiments suggest a folding mechanism with two parallel pathways via monomeric and dimeric intermediates with different cis/trans prolyl conformations and structural properties. The analyses of fluorescence anisotropy and far-UV CD measurements underline the complex folding behavior

Materials and Methods
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